Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the
SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson's disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5' untranslated region (5' UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5' UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound's selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.
Parkinson’s disease; RNA; chemical biology; intrinsically disordered proteins; α-synuclein.
Copyright © 2020 the Author(s). Published by PNAS.
Conflict of interest statement
Competing interest statement: M.D.D. is a founder of Expansion Therapeutics.
Design and characterization of an
SNCA 5′ UTR IRE-targeting small molecule. ( A) Schematic depiction of an α-synuclein –mediated disease pathway. ( B) Secondary structure of the 5′ UTR IRE of SNCA mRNA that regulates translation and the chemical structures of hit small molecules predicted by Inforna. ( C) Quantification of a Western blotting screen of candidate small molecules inhibiting α-synuclein protein expression in SH-SY5Y neuroblastoma cells. ( D) Cytoprotective effect of Synucleozid against α-synuclein toxicity in SH-SY5Y cells measured using a lactate dehydrogenase assay. Synucleozid abrogates cytotoxicity induced by α-synuclein preformed fibrils, which act as seeds and recruit endogenous α-synuclein to aggregate. * P < 0.05, ** P < 0.01, *** P < 0.001, as determined by ANOVA. Error bars indicate SD.
Synucleozid shows on-target effects in cells. (
A, Top) Structures of designed luciferase (Luc) reporter plasmids used in selectivity studies. ( A, Bottom) Luciferase assay of Synucleozid effects in SH-SY5Y cells stably transduced with plasmids containing the 5′ UTR of SNCA, amyloid precursor protein, prion protein, or ferritin mRNAs. ( B) Selectivity of Synucleozid for inhibiting α-synuclein protein translation as compared with its effect on APP, PrP, ferritin, and transferrin receptor determined by Western blotting. * P < 0.05, ** P < 0.01, *** P < 0.001, as determined by ANOVA. Error bars indicate SD.
Selectivity of Synucleozid for the A bulge in the
SNCA IRE. ( A) Secondary structure of 2-AP–labeled RNA used in the assays. ( B) Plot of the change in 2-AP fluorescence as a function of Synucleozid concentration. ( C) Plot of the affinity of Synucleozid for various SNCA IRE mutants as determined by competitive binding assays with the 2-AP–labeled RNA. RNA-0 is native SNCA IRE. RNA-12 is a fully base-paired RNA in which all 5 internal bulges and loops have been mutated. Each of RNA-1 to RNA-5 has 1 bulge or loop mutated. RNA-6 to RNA-12 are mutants of the A bulge or have mutated closing base pairs ( ). Error bars indicate SD. SI Appendix, Figs. S7 and S8
ASO-Bind-Map studies confirm that Synucleozid binds to the predicted site in vitro and in cells. (
A) Designed ASOs that tile through the SNCA IRE. ( B, Left) Scheme of RNase H-mediated ASO-Bind-Map. ( B, Right) Relative cleavage of full-length SNCA IRE by RNase H after hybridization of ASOs with or without Synucleozid preincubation. Statistical significance was calculated between each specific ASO with or without Synucleozid preincubation. ( C, Left) Scheme of FRET-based ASO-Bind-Map. ( C, Right) Normalized relative fold change of Cy5/Cy3 fluorescence for various ASOs with or without Synucleozid preincubation. ** P < 0.01, *** P < 0.001, as determined by a 2-tailed Student t test. Error bars indicate SD.
Cellular ASO-Bind-Map validates the
SNCA IRE as the target of Synucleozid. ( A) Scheme of ASO-Bind-Map studies completed in cells. ( B) Expression of SNCA mRNA in SH-SY5Y cells transfected with designed gapmers. Synucleozid protects SNCA mRNA from RNase H-mediated cleavage by ASO(1–10), which hybridizes with the Synucleozid binding site. Protection is not observed from cleavage mediated by ASO(29–39), which hybridizes to a distal site. * P < 0.05, ** P < 0.01, *** P < 0.001, as determined by ANOVA. Error bars indicate SD.
Investigation of a potential mode of action of Synucleozid. (
A) Synucleozid could affect the loading of SNCA mRNA into polysomes and/or the assembly of active ribosomes, which can be studied by polysome profiling. ( B) Representative absorption trace (at 254 nm) of polysome fractionation from polysome profiling of SH-SY5Y cells treated with Synucleozid (1 μM) or vehicle (dimethyl sulfoxide; DMSO) ( Top) and quantification of the percentage of SNCA mRNA level in each fraction relative to total SNCA mRNA expression, as assessed by RT-qPCR ( Bottom). ( C) Percentage of SNCA mRNA present within monosome- and polysome-containing fractions with (black) and without (white) Synucleozid (1 μM) treatment. Fractions labeled as “incomplete ribosome” contain 40S and 60S ribosomal subunits (fractions 1 to 5); “single ribosome” (fractions 6 and 7) indicates 80S ribosomes. * P < 0.05, ** P < 0.01, as determined by a 2-tailed Student t test. Error bars indicate SD.
Investigation of 2 other potential modes of action of Synucleozid. (
A) Synucleozid could affect the abundance of IRPs and/or the affinity of the IRP–IRE complex, which can be assessed by Western blotting and immunoprecipitation. ( B) SNCA mRNA was pulled down from treated (Synucleozid; 1 μM) and untreated (vehicle; DMSO) SH-SY5Y cells by immunoprecipitation of IRP-1 (black bars) or IRP-2 (white bars). SNCA mRNA levels were quantified by RT-qPCR. Iron (II) and deferoxamine are positive controls for IRP-1, and ASO is a positive control for IRP-2, each used to detect changes in the amount of immunoprecipitated mRNAs. The amount of SNCA mRNA bound to IRP-1 or IRP-2 shows no significant difference with or without Synucleozid treatment. * P < 0.05, *** P < 0.001, as determined by a 2-tailed Student t test. Error bars indicate SD.
Global proteome profiles of SH-SY5Y cells after treatment with Synucleozid or an siRNA directed at α-synuclein. (
A and B) Volcano plots of SH-SY5Y cells treated with ( A) α-synuclein siRNA vs. a scrambled control (0.1 μM), or ( B) Synucleozid (1.5 μM) vs. vehicle are shown. Data are represented as log 2 fold change; dotted lines represent a false discovery rate of 1% and an S 0 of 0.1, indicating an adjusted P value of 0.01. Red dots represent the common up-regulated proteins in the oxidative phosphorylation pathway. ( C) Venn diagrams showing down- or up-regulated proteins upon α-synuclein siRNA or Synucleozid treatment compared with their respective controls.
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